Lung infections are major causes of acute lung injury (ALI), with limited effective treatment available. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential adaptor regulating Toll-like receptors (TLRs). We recently identified Cullin-5 (Cul-5) as a prominent component in the regulation of TRAF6 polyubiquitination, but its physiological significance in acute lung injury has not been explored. In this study, we investigated the potential role of Cul-5 in regulating ALI using mice receiving intratracheal instillation of LPS. We observed that Cul-5 deficient mice displayed reduced lung injury compared with wild-type mice as evidenced by histological analysis, alveolar neutrophil infiltration and lung liquid accumulation. In addition, inflammatory cytokine expression in bronchoalveolar lavage fluid and lung tissue was also markedly reduced in LPS-treated Cul-5 deficient mice. Interestingly, intratracheal adoptive transfer of Cul-5+/- but not Cul-5+/+ macrophages attenuated neutrophil recruitment, alveolar inflammation, and loss of barrier function in LPS-challenged wild-type mice. Finally, we demonstrated that Cul-5 neddylation following LPS exposure induced Cul-5 and TRAF6 interaction and, thereby, TFAR6 polyubiquitination, leading to NF-B activation and generation of proinflammatory cytokines. Our data show that Nedd8 modification of Cul-5 is required for its interaction with TRAF6 and activation of the TLR4-TRAF6 signaling pathway in LPS-induced acute lung injury, a feature that may be explored for therapeutic intervention.